Project Summary/Abstract. The goal of this project is to test the hypothesis that perfluoroalkylated substances (PFAS) negatively impact formation of the placenta, and consequently, pregnancy outcomes. This work gains added significance in light of the increasing public health concerns towards these persistent compounds. The proposed experiments will also fill gaps in our understanding regarding the effects of these chemicals during human placental development, about which little is known. Pregnant mothers are exposed to a variety of chemicals, including PFAS. The latter exposures are widespread and high levels are linked with adverse effects on thyroid function, cholesterol metabolism, and birth outcomes. The placenta, a temporary embryonic/fetal organ that forms during pregnancy, facilitates gas, nutrients, and waste exchange with the mother. Deficiencies in placental development and function underlie numerous pregnancy complications, such as preeclampsia and intrauterine growth restriction. Despite its importance much remains unknown about the placenta, especially its role as a toxicological target. Here I propose studying PFAS effects on the organ's population of progenitor cells, termed cytotrophoblasts (CTBs), which establish the architecture of the maternal-fetal interface during pregnancy. To do so I will use an in vitro model of this process. Primary CTBs will be isolated and exposed to PFOA, PFNA, or GenX. The toxicological effects of these PFAS will be elucidated in two ways. First, using the CTB model, relevant effective concentrations of PFOA, PFNA, or GenX will be determined and a mass spectrometry-based approach will be used to determine their global effects at the level of the proteome (Aim 1). Second, honing in on levels relevant to public health exposures, the functional relevance of PFAS protein targets that could play hierarchical roles in placental development will be investigated by mimicking the observed chemical effects, e.g., up or down regulation (Aim 2). Thus, the results of these experiments will advance our knowledge about the human health effects of the compounds during a critical developmental window. Completing this study will advance the applicant's training in important new directions that are enabled by the expertise of his primary mentor, Dr. Susan Fisher: human placental biology and mass spectrometry- based proteomics analyses. Dr. Hao Chen will receive valuable input from his mentorship team, composed of experts in prenatal environmental exposures, bioinformatics, and reproductive biology. In collaboration with his mentors, Dr. Chen will develop critical skills that are required for a successful transition to an independent academic career in environmental health. This will be accomplished through a focused development plan consisting of didactic courses and close collaboration with his mentors. At the conclusion of this proposal, Dr. Chen will have led the first investigation of PFAS effects on CTBs and their function, providing insight into the impact of these chemicals towards developmental and reproductive health.